Journal: bioRxiv

Article Title: Transcription initiation defines kinetoplast RNA boundaries

doi: 10.1101/350256

Figure Lengend Snippet: (A) Mitochondrial localization of MTRNAP-TAP. Subcellular fractionation was performed with MTRNAP-TAP expressing cell line. Cyto, cell lysate depleted of membranes by centrifugation; crude mito, mitochondria and cell membranes; pure mito, mitochondrial fraction purified by centrifugation in Renografin density gradient. Protein profiles were visualized by Sypro Ruby staining and specific proteins were detected by immunoblotting. (B) Growth kinetics of MTRNAP-TAP cell line after induction with tetracycline. (C) Native molecular mass of the MTRNAP complex. Mock-and RNase I-treated cell lysates were separated on a 10 – 30% glycerol gradients. Peak fractions were resolved on native gel alongside with molecular mass markers. MTRNAP-TAP was detected by immunoblotting. The fraction 3,4 are the same fractions from . (D) Southern blotting analysis of maxicircle DNA fragments generated by focused sonication in mock-and formaldehyde-crosslinked cells. Low, medium and high intensity shearing was performed with Covaris M220 Focused Ultrasonicator as described in Star*Methods. High intensity was used to generate KAP-Seq libraries.

Article Snippet: For Northern blotting, 50 μg was treated with 5U of RNase-free DNase I (New England Biolabs) in 0.1 ml of manufacturer-supplied buffer for 30 min at 37°C.

Techniques: Fractionation, Expressing, Centrifugation, Purification, Staining, Western Blot, Southern Blot, Generated, Sonication

Journal: bioRxiv

Article Title: Transcription initiation defines kinetoplast RNA boundaries

doi: 10.1101/350256

Figure Lengend Snippet: (A) MERS1 RNAi knockdown impact on pan-edited mRNA. Edited and pre-edited forms of representative RPS12 mRNA were analyzed by Northern blotting. The short A-tailed and long A/U-tailed edited mRNA populations are indicated. RNAi was induced by adding tetracycline. MERS1 downregulation was verified by immunoblotting . (B) MERS1 knockdown effects on unedited mRNAs was assessed by Northern blotting. (C) Ribosomal RNA Northern blotting in MERS1 RNAi cells. (D) Guide RNA Northern blotting in MERS1 RNAi cells. (E) Isolation of UV-induced MERS1-and MERS2-RNA crosslinks. CLIP: MERS1 was immunoaffinity-purified with polyclonal antibody from parental and DSS1 DN cells. CLAP: TAP-tagged MERS2 was purified by affinity pulldown with IgG-coated magnetic beads. Parasites were mock-treated (−) and UV-irradiated (+). RNA was fragmented by RNase A and T1, radiolabeled, released from the crosslink (areas indicated by brackets), and sequenced. (F) MERS2 in vivo binding motif. The MEME algorithm was applied to predict the sequences enriched in MERS2-crosslinked RNA. (G) MERS2 CLAP and MERS1 CLIP (from parental and DSS1 DN cells) reads were aligned to a representative maxicircle region with overlapping mRNAs encoded on the same strand. (H) PPsome binding sites in the maxicircle. MERS1 CLIP and MERS2 CLAP reads from parental and DSS1DN cell lines were mapped to the gene-containing region. Annotated mitochondrial transcripts, read count scales and maxicircle coordinates are indicated. (I) Composite distribution of PPsome complex binding sites in mitochondrial mRNAs. The reads were aligned to unedited (blue) and fully-edited sequences (red). Read counts located 1000 nt downstream and 100 nt upstream of the 5′-end in each transcript were collected in 1 nt bin. The average coverage across all maxicircle genes was plotted.

Article Snippet: For Northern blotting, 50 μg was treated with 5U of RNase-free DNase I (New England Biolabs) in 0.1 ml of manufacturer-supplied buffer for 30 min at 37°C.

Techniques: Northern Blot, Western Blot, Isolation, Purification, Magnetic Beads, Irradiation, In Vivo, Binding Assay

Journal: bioRxiv

Article Title: Transcription initiation defines kinetoplast RNA boundaries

doi: 10.1101/350256

Figure Lengend Snippet: (A) Partial alignment of NUDIX motifs from trypanosomal, bacterial and human pyrophosphohydrolases. EC_RPPH; Escherichia coli RppH (WP_088540307.1), HS_DCP2; Homo sapiens Dcp2 (NP_001229306.1), HS_MTH2; H. sapiens Mth2 (NP_060753.1). Metal binding acidic residues are shown by arrows. (B) Pyrophosphohydrolase activity of purified complexes. The MERS1 and RESC complexes, and the ribosome were affinity-purified via indicated subunits and incubated with 5′ [γ- 32 P]-labeled RNA. Reaction products were separated by thin-layer chromatography along with those produced by RppH NUDIX hydrolase from E. coli . (C) MERS1 relative abundance in purified complexes. Mock-treated and RNAi knockdown cells were analyzed along with tandem affinity purified RESC and the ribosome. Cross-reactivity of polyclonal antibodies raised against 6-His tagged recombinant MERS1 with likewise tagged GRBC5 bait is shown by an asterisk. (D) Cell growth kinetics of MERS1 RNAi and conditional knock-in (KI) cell lines. RNAi was induced with tetracycline to downregulate endogenous MERS1. In the knock-in, the drug was withdrawn to suppress conditional MERS1-TAP expression in KI cells. One endogenous allele constitutively expressed functional (WT-KI), or inactive (mut-KI), MERS1 proteins while the other allele was disrupted . RNAi repression, and conditional (MERS1-TAP) and constitutive (MERS1-WT and MERS1-mut) expression were verified by Western blotting. (E) Effects of MERS1 enzymatic activity loss on pan-edited mRNA. Pre-edited and edited forms of RPS12 mRNA were detected by Northern blotting. The short A-tailed and long A/U-tailed edited mRNA populations are indicated. (dT), RNA was treated with RNase H in the presence of oligo(dT) 20-mer to remove short A-tails and long A/U-tails. (F) Effects of MERS1 enzymatic activity loss on moderately-edited cyb , and unedited CO1 mRNAs, and 9S and 12S rRNAs.

Article Snippet: For Northern blotting, 50 μg was treated with 5U of RNase-free DNase I (New England Biolabs) in 0.1 ml of manufacturer-supplied buffer for 30 min at 37°C.

Techniques: Binding Assay, Activity Assay, Purification, Affinity Purification, Incubation, Labeling, Thin Layer Chromatography, Produced, Recombinant, Knock-In, Expressing, Functional Assay, Western Blot, Northern Blot

Journal: bioRxiv

Article Title: Transcription initiation defines kinetoplast RNA boundaries

doi: 10.1101/350256

Figure Lengend Snippet: (A) Mapping 5′ termini of antisense transcripts. A 5′ RACE was performed in the parental, MERS1 RNAi and DSS1 DN cells. RNA was treated with 5′ polyphosphatase to capture mono-and triphosphorylated transcripts (+P), or mock treated. Positions of canonical mRNA adenylation sites are shown in the top panel. The 5′ RACE reads for antisense RNAs were aligned to maxicircle sequences. Read counts located 100 nt downstream and upstream of the mapped polyadenylation site in each transcript were collected in a 1 nt bin. Composite distribution of antisense RNA 5′ ends within annotated mRNA boundaries is shown by summation of coverage across all genes. The 3′ RACE-defined polyadenylation site is set as zero. Read scale and maxicircle coordinates are indicated. (B) Detection of non-coding RNAs transcribed as antisense to major strand-encoded RPS12 and MURF5 pre-mRNAs. Total RNA from MERS1 RNAi and DSS1 DN cells was analyzed by Northern blotting. (dT), RNA was treated with RNase H in the presence of oligo(dT) 20-mer to eliminate A-tails. Note unaltered migration patterns in RNase H/oligo(dT) sample. (C) Antisense RNA-controlled 3′ end definition in vitro . Active (WT) and inactive (DN) DSS1 exonuclease variants were isolated from mitochondrial fraction by tandem affinity purification. Reactions with 5′ radiolabeled single-stranded (ss) RPS12 mRNA fragment, or pre-assembled partially double-stranded (ds) RNAs, were terminated by adding Proteinase K. Products were resolved on polyacrylamide denaturing (upper panel) or native (lower panel) gels. FP, final degradation products (4-5 nt). (D) Same reactions as in (C) were performed with ND7 mRNA fragment.

Article Snippet: For Northern blotting, 50 μg was treated with 5U of RNase-free DNase I (New England Biolabs) in 0.1 ml of manufacturer-supplied buffer for 30 min at 37°C.

Techniques: Northern Blot, Migration, In Vitro, Isolation, Affinity Purification

Journal: Redox Biology

Article Title: Regulation of metastasis suppressor NME1 by a key metabolic cofactor coenzyme A

doi: 10.1016/j.redox.2021.101978

Figure Lengend Snippet: NME1-4 are identified by LC-MS/MS analysis as CoA-binding proteins. The normalised total spectrum count refers to the total spectra established for a single protein.

Article Snippet: The PVDF membrane was subsequently incubated with mouse anti-CoA primary antibodies (1:6000) or rabbit anti-NME1 primary antibodies (1:3000, Proteintech) at 4 °C overnight or 2 h at RT.

Techniques: Molecular Weight, Clinical Proteomics, Membrane

Journal: Redox Biology

Article Title: Regulation of metastasis suppressor NME1 by a key metabolic cofactor coenzyme A

doi: 10.1016/j.redox.2021.101978

Figure Lengend Snippet: h NME1 is CoAlated at its redox-sensitive Cys109 in diamide-treated HEK293/Pank1β cells. (A) Anti-CoA Western blot reveals extensive modification of cellular proteins by CoA in HEK293/Pank1β cells treated with 500 μM diamide for 30 min, when compared to untreated cells; (B) The LC-MS/MS spectrum of the peptide (GDFC109IQVGR) corresponding to NME1, containing CoA-modified cysteine (C+356) at residue 109 was obtained as described in Methods from HEK293/Pank1β cells treated with diamide. Fragment ions are coloured cyan and red for y- and b-ions, respectively. The asterisks (*) denote the loss of phosphoric acid (−98 Da) from the precursor and/or product ions that contained the CoA-modified cysteine residue. (C) The X-ray crystal structure of h NME1 (PDB: 2HVD) is shown. The nucleotide binding pocket is indicated by a red ellipse, catalytic histidine residue (His118) in yellow and cysteine residues (Cys4, Cys109 and Cys145) in orange. The Kpn-loop (residues 89–114) is coloured in cyan. (D) The multiple sequence alignment of NME1-4 from residues 89 to 120 is shown. The redox-sensitive cysteine 109 is shown in orange, and conserved catalytic histidine 118 in yellow. The Kpn-loop (residues 89–114) of NME is shown in cyan. The residue numbering is based on the amino acid sequence of NME1. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The PVDF membrane was subsequently incubated with mouse anti-CoA primary antibodies (1:6000) or rabbit anti-NME1 primary antibodies (1:3000, Proteintech) at 4 °C overnight or 2 h at RT.

Techniques: Western Blot, Modification, Liquid Chromatography with Mass Spectroscopy, Residue, Binding Assay, Sequencing

Journal: Redox Biology

Article Title: Regulation of metastasis suppressor NME1 by a key metabolic cofactor coenzyme A

doi: 10.1016/j.redox.2021.101978

Figure Lengend Snippet: NME1 is CoAlated in vitro and in cellular response to oxidative and metabolic stress. (A) In vitro CoAlation of recombinant h NME1. In vitro CoAlation of 100 μM h NME1 was carried out in the presence of 400 μM CoASH; 400 μM CoASSCoA; or 700 μM CoASH and 2 mM H 2 O 2 . After buffer exchange, approximately 2 μg recombinant protein from each reaction was separated on SDS-PAGE gel under reducing (with DTT) or non-reducing conditions (without DTT). Gels were immunoblotted with anti-CoA or anti-NME1 antibodies or stained with Coomassie Blue. (B, C) CoAlation of h NME1 overexpressed in E. coli is strongly induced by oxidising agents, but weakly under glucose deprivation. The expression of h NME1 in E. coli transformed with pET28a (+)- h NME1 plasmid was induced with 0.5 mM IPTG for 18 h at 25 °C. Then, bacterial cultures were treated with 2 mM diamide, 10 mM H 2 O 2 or 100 μM NaOCl (in M9 media) for 10 min, or incubated in M9 medium for 10 min. Total bacterial lysates (B) and Ni-NTA Sepharose pulled-down samples (C) were separated by SDS-PAGE gel under non-reducing condition and immunoblotted with anti-CoA antibody. Monomers (M), dimers (D) or oligomers (O) of NME1 have been indicated. The Ponceau-stained membranes show the loading control. The figures shown are indicative of three independent repeats. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The PVDF membrane was subsequently incubated with mouse anti-CoA primary antibodies (1:6000) or rabbit anti-NME1 primary antibodies (1:3000, Proteintech) at 4 °C overnight or 2 h at RT.

Techniques: In Vitro, Recombinant, Buffer Exchange, SDS Page, Staining, Expressing, Transformation Assay, Plasmid Preparation, Incubation, Control

Journal: Redox Biology

Article Title: Regulation of metastasis suppressor NME1 by a key metabolic cofactor coenzyme A

doi: 10.1016/j.redox.2021.101978

Figure Lengend Snippet: NME1 CoAlation in mammalian cells is induced by oxidising agents and glucose deprivation. (A,B) Anti-CoA and anti-NME1 Western blot analyses of affinity purified transiently overexpressed h NME1 from HEK293/Pank1β cells treated for 30 min with (A) 500 μM diamide, or (B) a dose-course of H 2 O 2 . (C) Anti-CoA and anti-NME1 Western blot analyses of affinity purified transiently overexpressed h NME1 from HEK293/Pank1β cells subjected to 20 h-glucose deprivation (Gluc depr.). (D) Anti-CoA and anti-NME1 Western blot analyses of total protein CoAlation in HEK293/Pank1β cells glucose deprived for 20 h. Ponceau stain shows equal loading of samples. Monomers (M), dimers (D), trimers (T) or oligomers (O) of NME1 have been indicated. All figures shown are representative of at least three independent repeats. The anti-NME1 Western blots represent the respective amounts of NME1 purified from each sample for analysis (A – C) .

Article Snippet: The PVDF membrane was subsequently incubated with mouse anti-CoA primary antibodies (1:6000) or rabbit anti-NME1 primary antibodies (1:3000, Proteintech) at 4 °C overnight or 2 h at RT.

Techniques: Western Blot, Affinity Purification, Staining, Purification

Journal: Redox Biology

Article Title: Regulation of metastasis suppressor NME1 by a key metabolic cofactor coenzyme A

doi: 10.1016/j.redox.2021.101978

Figure Lengend Snippet: CoAlation of h NME1 inhibits its NDPK activity. (A) Upon incubation with CoASSCoA, the NDPK activity of h NME1 WT is reduced by ~53%. In the presence of DTT, which reduces the mixed disulfide bond between CoA and h NME1, the NDPK activity is restored up to 84.8%. CoAlation of h NME1 C109A mutant shows no reduction in NDPK activity. In the absence of Cys109, CoAlation does not occur close to the active site and therefore, h NME1 maintains its NDPK activity. Data represent mean ± SEM from n = 2 or 3 experiments. p values were calculated using Šidák multiple comparison, ordinary one-way ANOVA test (****p < 0.0001, *p < 0.05, ns = not significant). (B) NDPK activity of h NME1 WT and C109A mutant was measured in the presence of increasing concentrations of reduced CoA (0, 2 and 4 mM). Results show that non-covalently bound CoA to both h NME1 WT and C109A mutant competitively inhibits their NDPK activity. The data are presented as a mean ± S.D. of at least three independent experiments. Data represent mean ± SEM from n = 2 or 3 experiments. p values were calculated using Šidák multiple comparison, ordinary one-way ANOVA test (****p < 0.0001, ***p < 0.0006). (C) Predictive model of CoA binding to NME1 under non-stressed and stressed cellular conditions. During non-stressed conditions, ATP binds to the active site of NME1 in order to initiate its NDPK activity. However, under cellular stress conditions, CoA may potentially bind non-covalently with its ADP moiety to the nucleotide-binding pocket of NME1; in this way, CoA may compete with ATP and other NTPs for the active site and possibly act as a competitive inhibitor for its NDPK activity. During oxidising conditions, CoA can also form a mixed disulfide bond with Cys109. This is followed by the accommodation of the 3′-phospho-ADP moiety of CoA within the nucleotide binding pocket of NME1. Covalent anchoring of CoA onto NME1 would force NME1 into an inactive state.

Article Snippet: The PVDF membrane was subsequently incubated with mouse anti-CoA primary antibodies (1:6000) or rabbit anti-NME1 primary antibodies (1:3000, Proteintech) at 4 °C overnight or 2 h at RT.

Techniques: Activity Assay, Incubation, Mutagenesis, Comparison, Binding Assay